Chonic renal damage is characterized by different morphological changes that show common findings in different renal diseases. In the glomerular compartment the major modifications are mesangium expansion and glomerulosclerosis and in the tubulointerstitial compartment the characteristic modifications are tubular atrophy, interstitial fibrosis and a reduction in the number of capillaries (1). The study of histological sections of renal tissue has been used to grade the above mentioned modifications with a semiquantitative scale, indicating whether the lesion is absent, mild, moderate or severe. Since 30 years ago it has been shown that tubulo-interstitial changes, especially tubular atrophy and interstitial expansion, are the best correlates with renal function (2, 3). Moreover, in different chronic glomerulopathies it has been shown that the degree of interstitial inflammation also correlates with renal function and with the prognosis of the renal disease (4, 5). Although during the 70’s and 80’s it has been supported that glomerular changes are poorly correlated with renal function, during the last decade it has been shown that the histomorphometric assessment of mesangium expansion is also correlated with renal function (6, 7).

In the case of kidney transplantation renal biopsies have been used to evaluate acute and chronic renal damage. On 1993 in order to grade and diagnose renal allograft biopsies an international schema, known as the Banff criteria, was published (8). A new version with slight modifications was reviewed on 1997 (9). On the other hand, on 1997 a new system to grade acute renal allograft rejection was published, known as the CCTT criteria (10). These two schemas for the diagnose and grade of acute rejection have been tested in order to known their specificity, sensitivity and reproducibility in a clinical setting. The CCTT criteria classifies acute rejection in three degrees leading to a reproducibility of 80% in the diagnosis of acute rejection. In the case of the Banff schema, reproducibility varies for the different lesions. For example, arteriolar hyalinosis has a reproducibility of only 25% while the diagnosis of acute rejection has a reproducibility of 50% since in this schema there were six degrees of acute rejection (11, 12). These schemas have been demonstrated to be very useful in the clinical setting, when the graft biopsy is done in order to characterize morphological modifications associated with graft dysfunction.

During the last years it has been proposed the use of protocol renal allograft biopsies to diagnose subclinical acute rejection and early chronic transplant nephropathy (CTN) (13). However, the incidence of these lesions have a wide range of prevalence in different studies. For example, the Winnipeg group has reported an incidence of suclinical rejection around 30% during the first six months after transplantation, while other groups have reported an incidence lower than 10% (14,15,16).

The presence of mild CTN in protocol biopsies performed in stable grafts has also an important variability. The Winnipeg group has reported an incidence of CTN at 6 months after transplant about 20%, our group has reported an incidence about 42% at 4 months, and even it has been reported an incidence as high as 80% at 3 months. It is important to notice that in these studies renal function at the time of biopsy was very similar. In the next table I summarize the most relevant studies with protocol biopsies done early after transplantation. In all but one case, biopsies were graded according to the Banff criteria.

Table 1. Different studies in early protocol biopsies. Results are expressed as mean ± standard error of the mean. Serum creatinine, glomerular filtration rate (GFR) and proteinuria of patients displaying CTN on the protocol biopsy are also shown.

# In this study biopsies were not evaluated according to the Banff schema, and were categorized according to the presence of a low or high degree of chronic changes.

In the above mentioned studies, the authors described the presence of relatively mild chronic lesions, usually classified as CTN grade I. It is important to notice that age-related chronic changes mimic those changes of CTN. All but one of the above mentioned studies were not controlled with a donor biopsy. Kuypers et al (18) described an incidence of donor chronic changes as high as 54%. Thus, they observed an incidence of new CTN around 26%. Our group has also studied sequential donor and recipient biopsies. In our case, we observed an incidence of mild CTN in 16% of donor biopsies and an incidence of new CTN around 23% (19).

Since chronic renal damage observed in protocol biopsies shows a closer relationship with allograft failure than serum creatinine, it has been proposed to use chronic renal damage as a potential surrogate measure of renal allograft survival (15, 18, 19). Thus, protocol biopsies may be used as a primary efficacy end-point in the case of prevention trials, or as a selection criteria to include patients in the case of a secondary intervention trial. Since in the case of protocol biopsies we are evaluating relatively mild chronic changes and these changes are not distributed homogenously in the kidney, it may be very useful to explore the utility and limitations of quantitative approaches.

In native kidneys it has been observed that the quantification of interstitial chronic renal damage shows a high correlation with renal function. The most widely employed measure was cortical interstitial volume fraction (Vvint/c) since it is the best correlate of renal function (20, 21, 22). A closer relationship between chronic renal damage and renal function was described in native kidneys using an automatic image analysis system based on texture analysis (23).

In the case of kidney transplantation few attempts have been made to evaluate the utility of quantitative measures. Vvint/c in donor biopsies has been correlated with recipient renal function during the first two years after transplant (24). Nicholson et al have explored the utility of the estimation of Vvint/c in protocol biopsies done at different times of follow up (25).They observed a relationship between Vvint/c and renal function only in the case of biopsies done six months after transplant but not in biopsies done earlier. A similar result was obtained when they analyzed the relationship between allograft failure and Vvint/c; grafts showing an Vvint/c greater than 25% at 6 months have a high risk of later graft failure.

Our group has also explored the utility of Vvint/c in protocol biopsies done three months after transplantation. In these study of a set of 98 biopsies we demonstrated a relationship between the presence of CTN and Vvint/c, but a cut-off of Vvint/c associated with later graft failure was not found (15). It is important to remark that interstitial expansion may be related not only with interstitial fibrosis but also with infiltration of interstitium by inflamatory cells and by interstitial edema. In a set of protocol biopsies we have explored the relationship between Vvint/c and acute and chronic inflamatory scores according to the Banff schema. We observed that in donor biopsies and in late biopsies Vvint/c is related with ci-score, while in biopsies done at 4 months Vvint/c is related with i-score (non published observations). Thus, the quantification of interstitial chronic renal damage in kidney transplant recipients required the separated quantification of interstitial fibrosis and interstitial inflammation. Recently, it has been shown in six-month protocol biopsies a close relationship between the interstitial area immunostained for collagen III and long-term graft function (26).

On the other hand, the number of interstitial infiltrating cells in kidney transplant recipients has been widely studied in a clinical setting. We have explored the usefulness of estimating the number of CD45 positive cells per mm² of the cortical tissue in protocol biopsies (15). In our study we have not evaluated biopsies showing acute rejection since the incidence of this diagnosis in our set of biopsies is very low. Then, we have evaluated normal biopsies, biopsies displaying CTN, and biopsies with borderline changes associated or not with CTN. Although biopsies with concomitant CTN and borderline changes have a higher number of CD45 positive infiltrating cells than normal biopsies or biopsies with CTN, we observed a high range of variation and, thus, it was not possible to find a significative difference (15). We have also described that in one year-protocol biopsies the degree of interstitial inflamation is lower than in earlier biopsies (27).

Thus, the concomitant quantification of interstitial fibrosis and interstitial infiltration may be useful to describe the presence of renal damage in renal transplant biopsies. Obviously, quantitative approaches will be less useful when acute and chronic damage are very high, since in this case semiquantitative scales may proportionate a similar information than quantitative measures and are easier to be obtained. By contrast, in the case of mild acute and chronic damage the quantification of acute and chronic lesions will allow to obtain more homogenous results by different groups, and this may be specially true in the case of protocol biopsies. Moreover, the quantification of renal damage may be done by completely automatic techniques(28) and, thus, measures of renal damage can be estimated with fully reproducible methods.

1. Tisher CC, Madsen KM. Anatomy of the kidney. In: The Kidney. Brenner BM i Rector FC, eds. WB Saunders Company. Philadelphia 1991.
2. Risdon RA, Sloper JC, de Wardener HE. Relationship between renal function and histological changes found in renal-biopsy specimens from patients with persistent glomerular nephritis. Lancet 1968; 2: 363-366.
3. Schainuck LI, Striker GE, Cutler RE, Benditt EP. Structural-functional correlations in renal disease. Part II: The correlations. Human Pathology 1970; 1: 631-641.
4. Alexopoulos E, Serón D, Hartley RB, Nolasco F, Cameron SJ. Immune mechanisms in idiopathic membranous nephropathy: the role of the interstitial infiltrates. Am J Kidney Dis 1989; 13: 404-412.
5. Alexopoulos E, Serón D, Hartley RB, Nolasco F, Cameron SJ. The role of interstitial infiltrates in IgA nephropathy: a study with monoclonal antibodies. Nephrol Dial Transplant 1989; 4: 187-195.
6. Vleming LJ, Baelde JJ, Westendorp RGJ, Daha MR, Van Es LA; Brunn JA. The glomerular deposition of PAS positive material correlates with renal function in human kidney diseases. Clin Nephrol 1997; 47 (3): 158-167.
7. Remuzzi A, Mazerska M, Gephardt GN, Novick AC, Brenner BM, Remuzzi G. Three-dimensional analysis of glomerular morphology in patients with subtotal nephrectomy. Kidney Int 1995; 48: 155-162.
8. Solez K, Axelsen RA, Benediktsson H, Burdick JF, Cohen AH, Colvin RB et al. International standardization of criteria for the histologic diagnosis of renal allograft rejection: The Banff working classification of kidney transplant pathology. Kidney Int 1993; 44: 411-422.
9. Racusen LC, Solez K, Colvin RB, Bonsib SM, Castro MC, Cavallo T et al. The Banff 97 working classification of allograft pathology. Kidney Int 1999; 55: 713-723.
10. Colvin RB, Cohen AH, Saiontz C, Bonsib S, Buick M, Burke B i cols. Evaluation of pathologic criteria for acute renal allograft rejection: reproducibility, sensitivity, and clinical correlation. J Am Soc Nephrol 1997; 8: 1930-1941.
11. Marcusen N, Olsen TS, Benediktsson H, Racusen L, Solez K. Reproducibility of the Banff classification of renal allograft pathology. Inter and intraobserver variation. Transplantation 1995; 60: 1083-1089.
12. Solez K, Marcusen N, Racusen L, Olsen TS. Reproducibility of the Banff classification of early renal allograft biopsies. J Am Soc Nephrol 1992; 3: 882-886.
13. Rush DN, Nickerson P, Jeffery JR, McKenna RM, Grimm PC, Gough J. Protocol biopsies in renal transplantation: research tool or clinically useful?. Current Opinion in nephrology and Hypertension 1998; 7: 691-694.
14. Rush DN, Jeffery JR, Gough J. Sequential protocol biopsies in renal transplant patients. Clinico-pathological correlations using the Banff schema. Transplantation 1995; 59: 511-514.
15. Serón D, Moreso F, Bover J, Condom E, Gil-Vernet S, Cañas C, et al. Early protocol renal allograft biopsies and graft outcome. Kidney Int 1997; 51: 310-316.
16. Kuypers DRJ, Chapman JR, O’Connell PJ, Allen RDM, Nankivell BJ. Predictors of renal transplant histology at three months. Transplantation 1999; 67: 1222-1230.
17. Dimény E, Whalberg J, Larsson E, Fellström B. Can histopathological findings in early renal allograft biopsies identify patients at risk for chronic vascular rejection?. Clin Transplantation 1995; 9: 79-84.
18. Legendre C, Thervet E, Skhiri H, Mamzer-Bruneel MF, Cantarovich F, Noël LH, Kreis H. Histologic features of chronic allograft nephropathy by protocol biopsies in kidney transplants recipients. Transplantation 1998; 65: 1506-1509.
19. Serón D, Moreso F, Ramón JM, Hueso M, Condom E, Fulladosa X, et al. Protocol renal allograft biopsies and the design of clinical trials aimed to prevent or treat chronic allograft nephropathy. Transplantation (in press).
20. Bohle A, Grund KE, Mackensen S, Tolon M. Correlations between renal interstitum and level of serum creatinine. Virchows Arch A Path Anat and Histol 1977; 373: 15-22.
21. Mackensen-Haen S, Bader R, Grund KE, Bohle A. Correlations between renal cortical interstitial fibrosis, athrophy of the proximal tubules and impairment of glomerular filtration rate. Clinical Nephrology 1981; 15: 167-171.
22. Eknoyan G, McDonald MA, Appel D, Truong LD. Chronic tubulo-interstital nephritis: correlation between structural and functional findings. Kidney Int 1990; 38: 736-743.
23. Moreso F, Serón D, Vitrià J, Grinyó JM, Colomé-Serra MF, Parés N, Serra J. Quantification of interstitial chronic renal damage by means of texture analysis. Kidney Int 1994; 46: 1721-1727.
24. Serón D, Carrera M, Griñó JM, Castelao AM, López-Costea MA, Riera L, Alsina J. Relationship between donor interstital surface and post-transplant function. Nephrol Dial Transplant 1993; 8: 539-543.
25. Nicholson ML, McCullogh TA, Harper SJ, Wheatley TJ, Edwards CM, Feehally J, Furness PN. Early measurement of interstitial fibrosis predicts long-term renal function and graft survival in renal transplantation. Br J Surgery 1996; 83: 1082-1085.
26. Nicholson ML, Bailey E, Williams S, Harris KPG, Furness PN. Computorized histomorphometric assessment of protocol renal transplant biopsy specimens for surrogate markers of chronic rejection. Transplantation 1999; 68: 236-241.
27. Serón D, Moreso F, Condom E, Carrera M, Cañas C, Cruzado JM, et al. Evaluation of interstitial lesions in well-functioning renal allografts. Transplant Proc 1995; 27 (4): 2213-14.
28. Serón D, Moreso F, Gratin C, Vitrià J, Condom E, Grinyó JM, Alsina J. Automated classification of renal interstitium and tubules by local texture analysis and a neural network. Analytical Quant Cytol and Histol 1996; 18 (5): 410-419.